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Abstract

Background

Variability in reported histopathology parameters in operated periampullary adenocarcinomas
may affect the prognostic weight of the parameters. Standardized axial sectioning
produces a higher incidence of involved margins and also seems to produce a lower
relative incidence of pancreatic compared with distal bile duct origin and a higher
incidence of involved lymph nodes, compared with non-standardized procedure. The aims
of this study were to 1) assess how a previously not described standardized pathology
procedure, with longitudinal sectioning along the distal bile duct, affects reported
tumour origin, margin status and involved lymph nodes, compared with non-standardized
procedure, 2) assess if re-evaluation of microscopic slides affects the prognostic
value of margin status and 3) compare the results of this standardized procedure with
reported results of other standardized and non-standardized procedures.

Methods

One hundred seventy-five consecutive pancreaticoduodenectomy specimens with primary
adenocarcinomas, operated during 2001 – 2011 at the University hospitals of Lund and
Malmö, Sweden, were re-evaluated histologically, and parameters relevant for classification
and prognosis were assessed, with 1 mm as a threshold for involved or uninvolved margins.
Follow-up lasted until 31 December 2013. Five-year overall survival (OS) and hazard
ratios (HR) were calculated for the margin status stated in the original reports and
margin status after re-evaluation.

Conclusions

Both the method of sectioning the specimen and the microscopic assessment affect prognostic
pathology parameters significantly. The results of the herein described standardized
method are similar to the results of other standardized procedures. The 1-mm threshold
for involved margins in pancreaticoduodenectomies is relevant for OS, and margin status
is an independent prognostic parameter.

Keywords:

Background

Pathology guidelines that change the incidence of histopathology parameters are clinically
relevant since the parameters carry prognostic information. Guidelines on gross examination
and sectioning of pancreaticoduodenectomy (PD) specimens have changed during the last
years, after the introduction of the Leeds pathology protocol (LEEPP) [1]. This standardized procedure raised the incidence of involved margins (R1) and involved
lymph nodes (N1), and also decreased pancreatic origin and increased distal bile duct
origin [2,3] compared to large series using non-standardized procedures [4-10].

Proportions of tumour origin vary greatly between different series of operated periampullary
adenocarcinomas and it is not known which proportions most accurately reflect the
biology of the tumours, or are most clinically relevant. It is however evident that
a meticulous pathology examination improves the quality of the pathology report for
these cancer forms by producing a higher incidence of N1 and R1 [2]. A high proportion of R1 also seems to correlate to a low relative incidence of pancreatic
origin, suggesting that a more thorough examination decreases the relative incidence
of pancreatic origin [11]. So far, the reported increase of R1 and decrease in pancreatic origin in the LEEPP-series
has been attributed to this particular slicing method. It is however not clear to
what extent this change is due to the method or to the interest and dedication of
the pathologist.

Here, we present the results of a different standardized protocol (SP), in which the
pathologist gains access to the full length of the common bile duct through a longitudinal
opening via the posterior margin of the PD-specimen, and only standard size blocks
are made. It has been stated that this method is inferior to the LEEPP, due to its
limited value for assessing tumour origin and resection margins [1]. This method has however not been studied in a standardized setting before.

Methods

Data collection and patient characteristics

The study cohort is a retrospective consecutive series of 175 PD-specimens with primary
adenocarcinomas surgically treated at the University hospitals of Lund and Malmö,
Sweden, from January 1 2001 until December 31 2011. Data on survival were gathered
from the Swedish National Civil Register. Follow-up started at the date of surgery
and ended at death or at December 31 2013, whichever came first.

Data on margin status was collected from the original pathology reports, as were data
on age at surgery, date of surgery, sex, and whether the specimen was handled according
to the SP or not. Data was also gathered on the origin of lymph nodes submitted in
separate containers. After information was given on how and from where the surgeons
harvested lymph nodes submitted in separate containers, positions 6, 8, 12, 13, 14
and 17 were classified as originating from the specimen, and other positions including
9 and 16 were classified as not originating from the specimen.

Of the 175 PDs, 46 (26%) were examined and sectioned according to our SP by one pathologist
(JE) and 129 (74%) were examined and sectioned by several pathologists according to
personal choice (non-standardized protocol, NSP).

Ethical permission was obtained from the Ethics Committee at Lund University.

Sectioning of the specimens, standardized protocol

This method is, by opening the PD-specimen along the bile duct, similar to one of
the methods earlier described by the Royal College of Pathologists [12] but performed in a standardized manner and without opening the pancreatic duct.

The specimens were handled after fixation in formalin (Figure 1). Margins were stained in different colours; one for the pancreatic transection margin,
one for the margin towards the superior mesenteric vein (SMV), one for the margin
towards the superior mesenteric artery (SMA), one for the anterior surface and one
for the posterior margin. The specimens were accessed through a longitudinal opening
of the common bile duct at the posterior margin, from the most proximal part of the
bile duct through the papilla of Vater. In the same plane the section was deepened
through the common bile duct and into the pancreatic parenchyme. This produced a book-like
opening that visualized the whole length of the common bile duct, the ampulla and
adjacent pancreatic parenchyme as well as parts of the posterior margin and parts
of the SMV-margin. Several standard size blocks were sampled from the ampulla with
adjacent duodenal mucosa, pancreatic parenchyme and anterior and posterior margins.
The bile duct was sampled longitudinally, with adjacent pancreatic parenchyme, posterior
margin and SMV-margin. Additional standard size blocks were sampled from the SMA-margin,
from all visible or palpable lymph nodes in the specimen and from additional areas
with possible tumour growth. En face sections were made from the pancreatic, bile
duct, pyloric and duodenal transection margins.

Figure 1.Accessing a pancreaticoduodenectomy specimen through the bile duct. (A) Anterior view of a painted pancreaticoduodenectomy specimen. Posterior – black, anterior
– blue, pancreatic transection margin – yellow, SMV-margin - red and SMA-margin -
green. A probe is inserted in the lumen of the bile duct. (B) posterior view, (C) posterior view with the bile duct opened longitudinally, and (D) the pancreatic parenchyme accessed through the bile duct, visualizing the ampulla,
the bile duct and parts of the pancreatic parenchyme, as well as parts of the margins.

Standardized protocol vs non-standardized protocol

Re-evaluations of slides

All haematoxylin & eosin stained slides from all cases were revised by one pathologist
(JE), blinded to the original report and outcome. Other stains were not revised or
used for the assessment of any parameter. Data were gathered on tumour origin, size
and grade, perineural invasion, lymphatic vessel and blood vessel invasion, invasion
of peripancreatic fat, number of lymph nodes and involved lymph nodes found by the
pathologist in the specimen, number of lymph nodes and involved lymph nodes harvested
from the specimen by the surgeon and submitted in separate containers, number of lymph
nodes and involved lymph nodes in separate containers originating from other areas,
N-stage, T-stage and margin status.

Decision on tumour origin was based on the anatomical centre of the tumour, with the
aid of preinvasive precursor lesions or multifocality, if present. A tumour in the
duodenal mucosa with intestinal morphology that involved the ampulla in the periphery
was considered to be of duodenal origin. A similar tumour with the ampulla in the
centre was considered to be of ampullary origin. A tumour along the bile duct that
involved the ampulla was considered to be of bile duct origin if the ampulla was in
the periphery of the tumour, but of ampullary origin if the ampulla was in the centre.
Multifocal tumour growth or multifocal premalignant changes in the pancreatic parenchyme
in the absence of evidence of other tumour origin was considered as a sign of pancreatic
origin. In addition to tumour origin the distinction between intestinal morphology
and pancreaticobiliary morphology was made for all ampullary carcinomas using morphological
criteria [13].

For the assessment of tumour grade, only the poorest degree of differentiation was
recorded.

Margin status was denoted as R1 if cancer was present less than 1 mm from any margin
except for the duodenal serosa, as R0 if the shortest distance exceeded 1 mm, and
as unknown (Rx) if any margin, except the duodenal serosa close to the cancer, was
insufficiently sampled. If a margin was considered sufficiently sampled or not differed
by the location of the tumour. In addition to pancreatic and distal bile duct transection
margins, an ampullary carcinoma needed at least one standard size block showing the
relation to the anterior surface, adjacent to the duodenal wall, two showing the relation
to the posterior surface adjacent to the duodenal wall, one from the SMA-margin and
one from the SMV-margin, in order to be considered sufficiently sampled regarding
margins. Carcinomas of pancreatic or distal bile duct origin needed, in addition to
pancreatic and distal bile duct transection margins, at least two blocks showing the
relation to the posterior margin, one from the SMA-margin, one from the SMV-margin
and one from the anterior margin. For duodenal origin, one block each from the posterior
and anterior margins adjacent to the duodenal wall was considered sufficient. A case
could be considered as R1 in an unspecified margin even if other margins were insufficiently
sampled.

For sampling of lymph nodes in the specimen, the full surface around the specimens
was searched manually and also visually after sectioning in intervals of approximately
3 mm.

Statistical analysis

The Chi-square test and Fisher’s Exact test were used to analyse differences in the
distribution of histopathological factors in relation to use of standardized vs non-standardized
protocol, and according to tumour location. Kaplan-Meier analysis and log rank test
were used to illustrate differences in 5-year overall survival (OS) in strata according
to margin status. Cox regression models were used to calculate hazard ratios (HR)
for the impact of histopathology parameters on 5-year OS, in univariable and multivariable
analysis, adjusted for age, sex, tumour morphology, tumour size, tumour grade, T-stage,
N-stage, margin status, perineural invasion, growth in peripancreatic fat, invasion
of lymphatic vessels and invasion of blood vessels. Cases who died within 1 month
from surgery (n = 2) or were lost to follow up (n = 1) were excluded from the survival
analyses.

All tests were two-sided and a p-value <0.05 was considered statistically significant.
All statistical analyses were performed using IBM SPSS Statistics version 20.0 (SPSS
Inc., Chicago, IL, USA).

Results

The annual PD-rate increased during the study period, with 35 and 29 cases operated
in 2010 and 2011, respectively, compared to a median of 13 per year (range 8–19) during
2001–2009. Forty-two of the 46 SP-cases were diagnosed during 2010 – 2011, which coincided
with an increased number of lymph nodes sent for analysis in separate containers;
median 1 (interquartile range, IQR 0 – 2) during 2001 – 2009 and median 7 (IQR 3.25
– 10) during 2010 – 2011.

Median 5-year OS was 30.4 months in the full cohort of all 172 SP- and NSP-cases,
35.0 months in the SP-group and 29.7 months in the NSP-group. In the SP-group of 46
cases, 27 died during follow up and 19 were censored at December 31 2013. Out of the
129 NSP-cases, 3 were excluded from the survival analysis, but included in all other
analyses. Of the remaining 126 cases, 88 died during follow up and 38 were censored
at December 31 2013.

Differences in the distribution of histopathological parameters between SP-cases and
NSP-cases

As shown in Table 1, there were several significant differences in the distribution of histopathological
parameters between the re-evaluated NSP- and SP-materials.

Tumour origin differed between the SP-group and the NSP-group (p = 0.040), with a
higher proportion of distal bile duct origin (39% vs 21%) and a lower proportion of
ampullary origin (26% vs 45%) in the former.

There was no significant difference between the SP-group and the NSP-group regarding
the number of lymph nodes found by the pathologist in the PD-specimens, but the number
of lymph nodes harvested from the specimen by the surgeon, as well as the total number
of lymph nodes originating from the PD-specimens, was significantly higher in the
SP-group compared with the NSP-group (p < 0.001 for both).

The number of involved lymph nodes in the PD-specimens was also significantly higher
in the SP-group as compared with the NSP-group (p = 0.001), and the number of involved
lymph nodes from the PD-specimens submitted in separate containers and total number
of involved lymph nodes originating from the specimens differed significantly. The
proportion of cases with involved lymph nodes (N1-N2) did not differ significantly
between the SP-group and NSP-group.

Since the increase in the number of lymph nodes harvested from the specimen by the
surgeon occurred in 2009, a separate analysis on lymph node-variables was performed
for the last 2.5 years of the study period (July 2009 – 2011). This revealed a significant
difference between the SP-group (n = 44) and the NSP-group (n = 31) in the number
of involved lymph nodes found in the PD-specimens by the pathologist (median 2.5 vs
1, p = 0.046). There were however no significant differences in the total number of
lymph nodes from the specimen (median 16 vs 12, p = 0.601), fraction of cases with
10 or more lymph nodes (89% vs 74%, p = 0.128) or fraction of cases with involved
lymph nodes (71% vs 65%, p = 0.622).

As further shown in Table 1, there was a significantly larger proportion of R1 cases (p = 0.002), tumours larger
than 20 mm (p = 0.008), perineural tumour growth (p = 0.035) and infiltration of peripancreatic
fat (p = 0.002) in the SP-group compared with the NSP-group. In contrast, infiltration
of blood vessels was more often found in the NSP-group (p = 0.004).

We also examined the involvement of different resection margins by tumour type (Table 2). Significant differences (R0 vs R1 and Rx) between the SP and non-SP groups were
found at the posterior margin (p = 0.001), the SMA-margin (p < 0.001) and the SMV-margin
(p < 0.001), and in tumours of distal bile duct origin (p = 0.006).

Effect of re-evaluations of slides

The distribution of histopathological characteristics in the total re-evaluated material,
stratified by tumour origin, is shown in Table 3. In the original reports there were 14 NSP-cases without information on margin status.
Re-evaluation of slides changed margin status for the NSP-group, increasing R1 from
45/115 to 60/129 and decreasing R0 from 70/115 to 12/129 (p < 0.001), and re-evaluations
also rendered 56 NSP-cases with unknown margin status (Rx). Re-evaluation of slides
rendered a non-significant increase of R1 in the SP-material, from 63% (29/46) to
76% (35/46) (p = 0.257).

Re-evaluations revealed lymph node involvement in 20% (14/70) of NSP-cases that were
N0 in the original report. This caused a non-significant change in fraction with involved
lymph nodes in the NSP-group, from 46% (59/129) to 57% (73/129) (p = 0.105). Re-evaluations
rendered no alterations in the fraction of involved lymph nodes in the SP-material.

Figure 2.Kaplan-Meier estimates of five-year overall survival in relation to margin status
before and after re-evaluation. Five-year OS in relation to (A) margin status stated in the original reports and (B) margin status upon re-evaluation of slides.

Table 4.Five year overall survival in relation to margin status in the original reports and
after re-evaluation

Table 5.Unadjusted and adjusted hazard ratios for death within 5 years in relation to re-evaluated
histopathology parameters

Discussion

This is, to our best knowledge, the first report on standardized longitudinal opening
and slicing of the common bile duct in the handling of PD-specimens with primary adenocarcinoma.

Our results confirm previous reports on standardized protocols in the pathology examination
of operated periampullary adenocarcinomas by showing that a 1-mm cut-off in the assessment
of margin status is relevant for overall survival, both in unadjusted analysis and
after adjusting for other histopathology parameters. Microscopic re-evaluation of
margin status revealed a larger proportion of involved margins than stated in the
original reports. Thereby, the prognostic value of uninvolved margins was increased,
regardless of other histopathology parameters. This suggests that a “guilty until
proven innocent”-approach towards margins in pancreaticoduodenectomies gives more
accurate prognostic information than the opposite approach. Moreover, survival in
the large group of cases with unassessable margin status (Rx) differed significantly
both from cases with uninvolved margins and from cases with involved margins, suggesting
that it is not appropriate to classify these cases as R0.

The more frequent finding of growth in peripancreatic fat and perineural tumour growth
in SP-cases compared to NSP-cases may be an effect of more extensive sampling in the
periphery of the tumour as well as along the bile duct and margins in SP-cases compared
with NSP-cases.

Tumour infiltration in blood vessels was more often found in NSP-cases than in SP-cases
(29% vs 9%), which may be due to an unintended more thorough search for evaluable
pathology parameters in SP-cases that had very little coverage on margins and lymph
nodes. This model of explanation suggests that the proportion of cases with tumour
infiltration in blood vessels in the NSP-group more accurately reflects the actual
percentage of infiltration in blood vessels. As a cautionary remark, the possibility
of a type I error, i.e. a false positive detection of significant differences between
the NSP-group and the SP-group, should also be considered, since a large number of
comparisons have been performed. A type II error, i.e. failure to detect the true
incidence of involved blood vessels in the SP-group, is also possible due to the relatively
small sample size in this group.

Comparisons of the incidence of involved margins between our SP-material, excluding
duodenal origin, and other standardized series show 78% R1 (32/41) in our SP-group
compared with 59% (32/54) and 61% (51/83) in the LEEPP-series [2,3]. The incidence of involved margins is often not comparable between SP-series and
NSP-series, due to a 0-mm definition of margin involvement, or lack of definitions
on margin involvement in NSP-series. The fraction of cases with involved lymph nodes
is however comparable, showing that non-standardized series [4-10] report involved lymph nodes in less than 60% of cases, compared to more than 70%
in our SP-group and in the LEEPP-series. If such differences are coincidental or actually
statistically significant, as well as their potential clinical significance, remains
unknown. In the present study, we were able to demonstrate a significantly higher
number of involved lymph nodes in the specimens in the SP-group compared with the
NSP-group, despite a temporal association between an increased number of lymph nodes
harvested from the specimens by the surgeons and the studied standardized protocol.

In our material the differences in tumour origin between the SP-group and the NSP-group
were significant. It is however not known if there are any clinically relevant differences
between the tumour origins of standardized and non-standardized series. It has however
previous been shown that the morphological distinction between intestinal and pancreatobiliary
morphology has prognostic implications, not only in ampullary adenocarcinomas, but
in all periampullary adenocarcinomas, regardless of tumour origin [14]. Moreover, while differences in the expression of cytokeratins and mucins according
to morphology have been observed in ampullary carcinomas [15], these differences seem to be less evident in series stratified solely by the anatomical
centre of the ampullary adenocarcinomas [16]. These findings suggest that morphological and molecular tumour characteristics have
a greater prognostic impact than the appreciated tumour origin.

Despite a very different approach to the specimen, the results on tumour origin, N-stage
and margin status in our standardized group are similar to the results of the LEEPP-series
[2,3] and to a lesser degree similar to the results of two other variants on standardized
protocols [17,18]. Whether or not our standardized protocol was more time consuming or more demanding
than the LEEPP, and thus inferior due to practical reasons, has however not been studied.

Conclusions

A 1-mm threshold for margin involvement is relevant for overall survival in operated
periampullary adenocarcinomas, regardless of tumour origin and other histopathology
parameters. Standardized protocols on sectioning of pancreaticoduodenectomy specimens
seem to increase the yield of adverse prognostic histopathology parameters compared
with non-standardized protocols. Standardizations in pancreatic pathology are needed
to decrease unjustifiable variability in pathology reports, both for the sake of the
treatment of individual patients and for the sake of future studies and clinical trials.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

JE conceived of the study, collected data, performed the statistical analyses and
drafted the manuscript. KJ participated in the design of the study, statistical analyses
and drafting of the manuscript. Both authors read and approved the final manuscript.

Acknowledgments

This study was supported by grants from the Knut and Alice Wallenberg Foundation,
the Swedish Cancer Society, the Swedish Government Grant for Clinical Research, Lund
University Faculty of Medicine and University Hospital Research Grants.

References

Verbeke CS: Resection margins and R1 rates in pancreatic cancer–are we there yet?